Beneficial sterols in selected edible insects and their associated antibacterial activities

Edible insects are increasingly gaining popularity as research reveals multiple benefits. However, the rediscovery of natural products from insects as medicinal agents has received limited attention. This study aimed at evaluating the diversity of sterols in extracts of nine edible insects and potential antibacterial activities. Dichloromethane extracts of these insects were analyzed using gas chromatography–mass spectrometry to identify important sterols, followed by evaluation of their anti-bacterial activities. Nineteen sterols were identified with the highest recorded in African fruit beetle [Pachnoda sinuata (47.37%)], crickets [Gryllus bimaculatus (36.84%) and Scapsipedus icipe (31.58%)]. Cholesterol was the most prevalent, except in black soldier fly (Hermetia illucens). Bioactivity revealed S. icipe as the most potent extract against Escherichia coli and Bacillus subtilis whereas G. bimaculatus was highest against Methicillin-susceptible Staphylococcus aureus 25923. These findings unravels the diversity of sterols in edible insects and their possible application in food, pharmaceutical and cosmetic industries.

Although there is paucity of information regarding the origin of phytosterols in invertebrates, certain insects are exceptional since they are known to acquire them from their dietary sources as shown by desert locust 27 . However, B. mori larvae, which feeds mainly on mulberry leaves (Molus alba), only portrayed cholesterol and cholest-4-en-3-one as the present sterols (Fig. 6).
Antimicrobial effects of the sterol containing extracts. Antimicrobial activity tests were performed on the DCM fraction of the different insect extracts. The comparison of the inhibition zones of the extracts to the positive control revealed that they exhibited varying inhibitory zones. When the DCM dissolved samples were exposed to B. subtilis, S. icipe exhibited an appreciable inhibitory zone of 8.33 ± 0.58 mm as the highest amongst all the extracts of the extract from S. gregaria portrayed moderate inhibition zones (7.0 ± 0.58 mm) whereas R. differens and G. Krucki had the least inhibitory effects (6.33 ± 0.58 mm) against B. subtiilis. When the cricket extracts were subjected to E. coli, the inhibition zone of G. bimaculatus was determined to be 6.67 ± 0.58 mm whereas S. icipe exhibited no appreciable activity. The H. illucens extract (containing 98.4% fatty acid content) was the most active against E. coli exhibiting 8.0 ± 1.00 mm as the inhibition diameter. On the contrary, B. mori extract with 98.2% fatty acid content exhibited moderate inhibitory diameter against all the test pathogens. A comparison of the antibacterial activity was carried out by dissolving the sample in 20% of acetonitrile and subjecting to the test organisms. Markedly, S. gregaria had a 8.67 ± 0.58 mm inhibition zone against B. subtilis, while H. illucens the second highest had 7.67 ± 0.58 mm inhibition when exposed to B. subtilis pathogen. The exposure of E. coli to the extracts proved that P. sinuata was the most active with 7.67 ± 0.58 mm inhibitory effects. The extracts of R. differens and B. mori however, did not exhibit any significant antibacterial activity against E.coli. On the other hand, S. icipe, was the least active extract, with no discernible inhibition zone against B. subtilis and S. aureus organisms. It however indicated a 7.0 ± 1.00 mm inhibitory activity against E. coli (Table 2). Thus, the results appear to be consistent regardless of the solvent used in dissolving the extracts.
From the MBC results, it is evident that S. icipe extract proved to be the most active followed by G. bimaculatus. Macrotermes sp. showed moderate inhibition concentration (2.5 mg/mL) and R. differens showed the least activity against all the test organisms. Analysis of the activity across individual test organisms reveal that G. bimaculatus was more potent against S. aureus, S. icipe and G. Krucki against E.coli. The most potent extract against B. subtilis was found to be S. icipe. All the extracts exhibited no growth at a concentration of < 0.312 mg/ mL (Table 3). Indeed, in general lower MBC values were recorded against E. coli (< 0.312-2.5 mg/mL). This indicates that E. coli was more susceptible to the active components in the extracts than the other test organisms. This study provides an insight into the value of insects and their chemical components such as lipids.

Discussion
The evaluation of sterol richness and composition in edible insects revealed cholesterol to be the most abundant in majority of the samples. This could be attributed to its lipophilic nature and significance in the structural makeup of the cell membrane in living organisms thus modulating fluidity 24 . On the contrary, its reduced quantity in H. illucens could be due to the elevated fatty acid content, which might have obscured its biosynthesis since they have a common starter building block unit i.e. acetyl -CoA. The presence of stanols in P. sinuata is attributed to the action of the hydrogenase enzyme in the insect's body/gut. The minor structural variations between sterols www.nature.com/scientificreports/ and stanols may have a distinct impact on their functions and metabolisms. Moreso, their biotransformation could be related to the differences of individual phenotypes and the composition of gut microbiota present in the insects 28 .
The higher quantities of campesterol/γ-ergostenol, stigma-7-en-3β-ol (5α, 24S), and 24-propylidenecholest-5en-3β-ol in the H. illucens extract could be linked to the presence and the action of oxidoreductases on the sterol side chain 29 . Among these sterols, Giner et al. 29 found out that 24-propylidenecholest-5-en-3β-ol was produced in about 17 species of marine algae as a novel sterol. These findings are supported by the work by Vidal et al. 30 where oxidoreductases are named as key and most abundant enzymes in the catalysis of approximately one-third enzymatic activities found in BRaunschweig Enzyme Database (BRENDA). www.nature.com/scientificreports/ Cholest-4-en-3-one and cholesta-3,5-diene metabolites found in G. bimaculatus, S. icipe and B. mori are known to be transformed products from cholesterol. In particular, cholest-4-en-3-one is an intermediate product of the transformation process of cholesterol to coprostanol under anox conditions via oxygenase-independent reactions as established in bacteria 31 . On the contrary, cholesta-3,5-diene is a sole primary product when cholesterol is subjected to high temperatures > 300 °C.
The analysis of B. mori extract showed an incomplete profile from that depicted from mulberry leaves. The mulberry leaves have been documented to possess the following phytosterols: cholesterol, stigmasterol, sitosterol and campesterol 32 . The identification of the two sterols from B. mori as shown by this study could suggest that the dietary sterols in mulberry may have been converted into cholesterol and cholest-4-en-3-one depending on the larval stage investigated 33 . Thus, the molecular conversion of phytosterols and the metabolism of B. mori larvae remains to be fully elucidated and understood.
Furthermore, termites primarily depend on wood to obtain cellulose and nutrients that they need for survival. However, it is noted that sterol composition is crucial for cellulose biosynthesis as it is linked to cell wall formation 34 . As a result, more research should be directed towards determining all the sterols generated from cholesterol modifications employing various sterolomic approaches 35 .
This study showed an elevated fatty acid content (98.4%) in H. illucens extract (Fig. 8) which may be attributed to the large inhibitory zone observed. According to literature, the fatty acid content of H. illucens is estimated to be 30% most of which are categorized as antimicrobial lipids 36,37 . However, the B. mori extract with 98.2% of the fatty acid content exhibited moderate activity. It is imperative that further studies be carried out to ascertain the    38 , stigmasterol was found to be the main compound responsible for the observed larvicidal activity against C. quinquefasciatus and A. aegypti. Moreover, the high activity observed in S. icipe and G. bimaculatus could be attributed to the presence of 27-Nor-ergosta-5,22-dien-3-ol(3β, 22Z). Previous studies have shown that ergosterol derivatives have the potential to exhibit antibacterial, antitumor, cytotoxic, rheumatoid arthritis and even immune promoting properties 39 . For instance, when ergosterol and cholesterol were combined with aminoglycosides and tested against multiresistant bacterial strains, the activity of the aminoglycoside increased with higher sub-inhibitory concentrations of the sterols 40 .
Additionally, taraxasterol that was present in G. bimaculatus has been reported in literature to possess many important pharmacological actions that include anti-cancer, anti-allergic, anti-oxidant, and anti-inflammatory activities [41][42][43][44] . Therefore, it may be responsible for the enhanced antibacterial activities of G. bimaculatus (9 mm) against MSSA 25923 in comparison to S. icipe (6 mm). These results are in line with a study from which twelve triterpenoid substances, including taraxasterol, were isolated and purified from Mexican Asteraceae plants. Only taraxasterol molecule was found to have antibacterial activity against S. aureus 45 .
It is therefore important to understand the plausible biosynthesis of the identified sterols. There are three key phases in the production of (C-30) sterols starting from squalene as delineated in literature 5 . The first stage entails six steps that include: 1. The conversion of acetyl CoA to acetoacetyl CoA mediated by the enzyme acetoacetyl CoA thiolase (AACT).
The two-phosphorylation events at MVA's C-5 and a decarboxylation/elimination step changes MVA into IPP in the first stage; IPP, the basic C-5 building block, that is then added to the prenyl diphosphate co-substrates to generate longer chains.
The condensation reaction is repeated in the second stage with the addition of Δ 3 -IPP, yielding the C-15 allylic product farnesyl diphosphate. By the action of squalene synthase (SQS), two molecules of farnesyl diphosphate condense tail to tail into the C-30 acyclic polyene squalene. A NADPH-dependent mono-oxygenase reaction catalyzed by squalene epoxidase (SQE) converts the C-30 symmetric olefin to S-oxidosqualene, which is then cyclized by an oxidosqualene sterol synthase to generate the steroidal backbone structure as represented in lanosterol (Fig. 9). Lanosterol is transformed to cholesterol in the third stage. Conversely, the cycloartenol synthase (CAS) pathway is thought to be mostly a plant sterol pathway converting oxidosqualene to cycloartenol 14 . The enzymatic activities of sterol methyltransferases (SMT), which catalyze the methylation reactions at the (C-24) carbon atom in the side chain, are used to elucidate the mechanisms of variations in the ratio of molecular kinds of sterols such as campesterol and β-sitosterol 46 (Fig. 9).
Alternatively, it is postulated that cholesterol in insects can be synthetized via the enzymatic conversion pathway from β-Sitosterol. Here, β-sitosterol is first converted to fucosterol then to 24,28-epoxyfucosterol and www.nature.com/scientificreports/ desmosterol as intermediates. With the action of 24-reducing enzyme on desmosterol as a rate-limiting step, cholesterol is formed 33 .
In conclusion, we herein describe the first comparative study of sterols in edible insect extracts and indicate their potential antibacterial effects. The range of sterols identified in the various extracts were between 2 and 9 different types. Cholesterol was the most abundant sterol in all the extracts except in H. illucens. Extract obtained from P. sinuata portrayed an array of phytosterols as well as stanols. The sterol 24-propylidenecholest-5-en-3β-ol, www.nature.com/scientificreports/ which has been widely identified in green algal species, was only found in H. illucens extract. On the other hand, taraxasterol (known to possess anti-cancer, anti-allergic, anti-oxidant, and anti-inflammatory activities) was identified in G. bimaculatus extract. The extracts from the evaluated insects showed significant inhibitory activities against two clinically important (Methicillin-susceptible S. aureus 25923, E. coli 25922) and one indicator (B. subtilis) pathogens. On this background, products containing sterols from edible insects could be utilized as targets for drug discovery against disease causing pathogens. It is therefore recommended that further studies on the isolation of individual sterols from the insects be carried out to investigate their antibacterial effects. The potent phytosterols could be used to formulate products that can help in improving health status of people living in low and middle-income countries. Moreover, structure activity relationship (SAR) studies could be carried out on the different bioactive phytosterols (in line with the biosynthetic pathway) to improve the observed activity. Lastly, varying the rearing or diet conditions of these insects is suggested to improve their mass production and increase biodiversity of the sterols as a sustainable source.

Materials and methods
Materials. All  Insect extract preparation. Before commencement of the experiment, each insect sample was properly cleaned to remove the debris. The samples were then placed in an oven at 60 °C for at least 48 h. The dried insects were ground to obtain fine powder using a blender. Approximately 10 g of each ground sample were extracted with 80% methanol and evaporated in vacuo. To the residual aqueous phase, about 50 mL of distilled water was added and partitioned with equal volume of n-hexane to remove the fatty acids. This was followed by subsequent extraction using equal volume of DCM. The DCM soluble extract was concentrated in vacuo and the sample prepared for GC-MS analysis by making a concentration of 100 ng/μL in triplicates. Analysis of the sterols. Mass spectral data and retention times were compared with that of cholesterol standards and reference spectra published by library-MS databases, including National Institute of Standards and Technology (NIST) 08 and 11, to identify the sterol components. The matching level of quality for the identification of the sterols was taken to be ≥ 90% with exception of a few considered above and below 70% to be traces as indicated in the table. The content of fatty acids and sterols were calculated from the relative peak area of all the detected peaks and a percentage calculated thereafter. Inhibitory assays. The disk-diffusion assay was performed in sterile Mueller Hinton agar (MHA) medium prepared in separate sterile petri dishes; 90 mm in diameter (F&S Scientific, Nairobi, Kenya), and 25 mL was poured to each plate as described by Hudzicki 48 . From the overnight microbial cultures prepared as mentioned above, 100 μL from each bacterial species was spread uniformly using sterile beads, on separate petri dishes. Sterile 6 mm discs were placed onto each agar plate (including 2 other discs for the positive and negative control). To the disc, 20 µL of the sample solutions (10 mg/mL and 1 mg/mL for the positive control) were added, before the dishes were incubated for 24 h at 37 °C.

GC-MS instrument conditions.
All the extracts were subjected to Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) against the S. aureus, B. subtilis, and E. coli, following published protocols with minor modification 49 . MIC assays were conducted in 96 well microtiter plates in a serial dilution, ranging from 5, 2.5, 1.25, 0.625 and 0.313 mg/mL per extract using Mueller Hinton broth (MHB). First, 40 µL of MHB was pipetted into the wells, then 40 µL of each extract at a higher concentration (i.e. 10 mg/mL for a well of 5 mg/mL), in 5% DMSO was dispensed into respective wells. Finally, 10 µL of the test bacteria in autoclaved distilled water at 1.0 × 10 8 CFU/mL (OD = 0.132 at 630 nm) were dispensed in all the wells using a pipette, before the plates were covered with sterile lid and incubated for 24 h at 37 °C in an incubator shaker. Streptomycin (1 mg/mL) was used as the positive control, while 5% DMSO was used as the negative control. After incubation, 20 µL from wells with no turbidity were plated out on Mueller Hinton agar plate and was incubated for 24 h at 37 °C. The least concentration that showed no visible growth was taken as MBC. Triplicate experiments were conducted. Data analysis. The data obtained from the GC-MS was analyzed using the MSD ChemStation Data Analysis Application software equipped with Adams2, Chemecol and NIST11 database libraries. The chromatograms were illustrated using a graphical design software (Adobe illustrator CS2). One-way ANOVA statistical analysis was done using the R software version 2022.

Ethical approval. Institutional Review Board Statement:
The Authority to conduct the experiments and collect data was in accordance with the animal welfare regulations and granted by National Commission for

Data availability
The datasets generated from GC-MS and analysed during the study are included in this paper.